How the ribosome can use over twenty chemically distinct aminoacyl-tRNA substrates within a single catalytic apparatus to synthesize proteins remains a fundamental biological question. The original adaptor hypothesis states that aa- tRNA substrate specificity comes entirely from the interaction of the tRNA anticodon with the mRNA codon. Recent studies, however, have revealed that features of the tRNA adaptor well beyond the anticodon also play critical roles in regulating aa-tRNA selection; due to its assumed silent role, the contribution of the amino acid component of the aa-tRNA to substrate selection remains virtually unexplored. Despite this, the hypothesis that the translational machinery is blind to the amino acid is at odds with the growing number of unnatural amino acids that are poorly incorporated using misacylated tRNAs; even subtle perturbations to the chemical structures of the natural amino acids can dramatically arrest protein synthesis. Thus, we hypothesize that, contrary to the adaptor hypothesis, the ribosome is exquisitely specific not only for the tRNA adaptor, but also for the structure and electrostatics of the amino acid covalently attached to the tRNA. Here, we propose to test this hypothesis by determining which step(s) in the translation cycle exclude backbone analogs, charged side-chain analogs, and large side-chain analogs of the wt amino acid substrates. The results of these studies should broadly impact efforts to engineer the unnatural aa-tRNA and the translational machinery to expand the range of analogs that can be incorporated using misacylated tRNAs and our fundamental understanding of the role of the aa-tRNA itself in regulating the conformational transitions that underlie protein synthesis. PUBLIC HEALTH RELEVANCE: The long term goals of this research are (1) to engineer the translational machinery for the incorporation of biophysical probes into proteins directly as they are being synthesized in the cell and (2) to gain a fundamental understanding of the mechanism of protein synthesis by the ribosome. Just as biophysical methods for studying biomolecules in vitro have significantly impacted our fundamental understanding of biomolecule structure and function and ability to develop effective therapeutics for human disease, the ability to image protein networks in living cells by direct incorporation of unnatural amino acid fluorophores and other biophysical probes has the potential to make broad, significant contributions to our understanding of the mechanism of biological pathways and human disease. Because protein synthesis by the ribosome is a major cellular pathway, fundamental understanding of this pathway significantly impacts our ability to develop antibiotics and other classes of therapeutics based on their ability to perturb this pathway both in prokaryotes and eukaryotes.